Method of Determining Hypoxia

ABSTRACT

A method of determining hypoxia in fetal scalp blood sampled during labor including the determination of total lactate dehydrogenase (LDH) in plasma obtained from the sample. The method can include the additional determination of K, Mg, Ca, AST, ALT, lactate in the plasma and/or blood. Increased values of one or more of LDH, Mg, Ca, AST, ALT, lactate are indicative of hypoxia in the fetus. Also disclosed is the use of a plasma separation apparatus in the method.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.12/101,470, filed Apr. 11, 2008, which is a continuation-in-part ofApplication No. PCT/SE2007/050738, filed Oct. 12, 2007, which claimspriority to Swedish Application No. 0602158-8, filed Oct. 13, 2006, bothof which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a method of determining hypoxia. Oneaspect of the present invention relates to methods of determininghypoxia in fetal scalp blood during labor indicative of a risk of fetalorgan dysfunction. In another aspect, the present invention relates to adevice for determining hypoxia.

BACKGROUND OF THE INVENTION

Acute perinatal asphyxia, that is, hypoxia (insufficient oxygensaturation of fetal blood) during or close to birth, remains animportant cause of neurological damage in form of hypoxic-ischemicencephalopathy (HIE) in newborn infants. It is seen in 2-9/1000 terminfants and is followed by cerebral palsy (CP) and death in the severecases. In a global perspective about 4 million newborn children die eachyear and about 23% are caused by acute perinatal asphyxia. Due to a lackof resources undeveloped countries are worse off, but as understood bythe large numbers also in the western world this is a serious problem.Sweden can be seen as a representative country for the western worldwherein asphyxia will occur in about 7/1000 term births leading to2/1000 children being born with HIE. To prevent persisting damage causedby perinatal asphyxia it is important to detect hypoxia in a fetus asfast as possible upon its onset. Fast detection allows one to makedecisions about whether to intervene at a stage where persisting damagehas not occurred. The intervention substantially consists in bringingthe infant out as quickly as possible by instrumental delivery, inparticular by caesarian section.

Detection of acute perinatal asphyxia is presently done by monitoring offetal heart rate followed by measurement of pH or lactate measurement infetal scalp blood sampled through the vagina if an ominous fetal heartrate pattern is seen.

pH and lactate are indicators of metabolic acidosis caused by a switchto anaerobic metabolism in situation of insufficient oxygen supply. Inan oxygen starved fetus pyruvate is metabolised to lactate and energy.At present measurement of pH is the golden standard. The fastdetermination of pH however requires about 35 μl of scalp blood, whichis not easily obtained. Failure in the first determination is quitecommon (20%) as some studies indicate. Lactate is easier to measuresince only 5 μl of blood is needed and the analysis can be carried outat bed side. Lactate analysis can be carried out within one minute andthus is sufficiently quick.

Lactate and pH are also indicators of acute asphyxia. As such Lactateand pH can provide an indication of a totally healthy fetus subjected toa sudden acute onset of hypoxia-ischemia during birth. A significantproportion of all infants developing hypoxic ischemic encephalopathyhave had episodes of hypoxia-ischemia before entering the deliveryphase. They are more vulnerable to hypoxia-ischemia during birth and donot respond in the same way as healthy fetuses and therefore thecurrently used methods are not sufficient for this group of patients.

A method for monitoring childbirth comprising the measurement of lactatein fluids, such as vaginal fluids, is disclosed in WO 2005/034762 A1.Preliminary results from a recent Swedish randomized study of pH andlactate in fetal blood at partum show that lactate, as an indicator ofacidosis, is as good as pH. Neither lactate nor pH are ideal predictorsof moderate/severe HIE: the sensitivity is only 67% for lactate and 50%for pH, whereas the specificity is about the same, 76% for lactate and73% for pH. Also, the sensitivity and specificity in predicting acidosisin newborns are less than 70 percent for both lactate and pH. A recentSwedish report concludes that even in fetal monitoring by a combinationof cardiotography (CTG) and STAN (analysis of the cardiographic STsegment) there is a risk of not detecting perinatal asphyxia of a kindthat may result in encephalopathic damage (SBU Alert-rapport nr2006-04).

Enzymes known to be elevated in newborn infants subjected to asphyxiaduring labor are LDH (lactate dehydrogenase), ALT (alanineaminotransferase) and AST (aspartate aminotransferase) also known asliver enzymes. LDH is found in most of the cells in the body, and it isconsidered an unspecific enzyme. Therefore it is infrequently used inclinical work. Previously LDH was used as a marker of myocardial damagebut has now been replaced by more specific tests. AST and, inparticular, ALT are more specific to liver damage. In a study on theeffect of labor and delivery on plasma hepatic enzymes in the newborn ofa group of low-risk Chinese women LDH (lactate dehydrogenase), ALT(alanine transaminase), AST (aspartate transaminase), GGT (γ-glutamyltransaminase) were determined and correlated to maternal and neonatalcharacteristics (Mongrelli M et al., J Obstet Gynaecol Res, 26(1):61-63, 2000).

If a fetus is subjected to hypoxia during or close to birth the bloodflow in its body will be redistributed from “less important organs”(kidneys, liver, fat and gut) in favor of the brain, heart and adrenals.This tends to damage cells in unprivileged organs. Cell damage resultsin a leakage of enzymes, which enter circulation. If the hypoxia issevere cells will die, and enzyme concentration increase even more inthe blood. The rate of decline of LDH, AST and ALT (between 12-36 hours)makes it possible to detect cases more vulnerable to hypoxia-ischemiaduring delivery due to previous hypoxia started before the delivery.Hypoxia also affects the balance of electrolytes in the fetal body. Oneexample is the influx of calcium from the blood in to the cells duringhypoxia. It is shown that the concentration of serum ionized calcium isincreased during hypoxia in a newborn animal model. Calcium alsopredicts outcome (brain damage or not) in human infants. Other enzymesand electrolytes of interest that are changed during hypoxia in newbornmammals are potassium (K⁺), magnesium (Mg²⁺), sodium (Na⁺), glucose,creatinine kinase (CK) and GGT.

In a global perspective about 4 million newborn children die each yearand about 23% of these deaths are caused by acute perinatal asphyxia.The pathological mechanism of hypoxia-ischemia leading to injuries ofthe neurons in the brain is biphasic starting with the primary phasedirectly after birth. If an infant is successfully resuscitated thisprimary phase will be followed by a free interval that continues forhours. In two out of seven asphyxiated infants this free interval willbe followed by a secondary energy depletion resulting in delayed celldeath in the child's brain and a clinical picture with seizures (alsoknown as HIE). The free interval offers a possibility to minimize thedelayed cell death by hypothermia treatment (cooling the child's brainto 34.5° C.). Currently, however, there is no reliable means forpredicting which child will develop HIE and therefore have benefits fromhypothermia treatment.

In addition to during or close to birth, hypoxia is also a seriousconcern in many other medical conditions. For example, colorectal canceris one of the most common tumors in both genders, the incidence of whichis increasing every year. The current treatment involves a surgicalprocedure whereby the tumor is removed together with a radical part ofthe bowel. In the majority of these cases the distal and the proximalends of the bowel are thereafter put together again. This is referred toas an anastomosis. During this procedure, arterial blood supply to thepart of the bowel where the tumor is located is interrupted when thearterial vessels are cut. Post-operative complications due to leakage ofthe anastomosis can be anticipated in 7-10% of all operations. In suchcases, the contents of the bowel will leak out in the abdomen and causeinflammation, peritonitis, sepsis and potentially death. The main reasonfor this complication is insufficient blood supply to the area for theanastomosis (e.g. hypoxia-ischemia) as a result of the extirpation ofthe vessels. The current solution is to simply re-operate. Undesirably,it is currently not possible, during surgery, to foresee if theanastomosis will leak or not.

Other areas where hypoxia is a major concern include vascular surgeryand liver transplant surgery. For instance, a major factor determiningmorbidity and mortality in patients after liver transplantation therapyis preservation injury of the hepatic grafts (Leemaster 1997). LDH, ASTand ALT leakage into the perfusate is a measure of loss of the membraneintegrity of the liver cells (Kebis 2007).

One major shortcoming of prior devices and methods for determininghypoxia is that LDH can only be analyzed in plasma or serum. Further,they require a minimum of 150 microliters of whole blood for such ameasurement. Such volume of blood is difficult to receive from smallanimals, specific tissue or the unborn child during birth. Anotherproblem is that LDH is also present in red blood cells and haemolysis(rupture of red blood cells) will lead to false high values (beyonddetection limit).

Thus, there is a need for a method of analyzing LDH, alone or togetherwith electrolytes and liver enzymes, quickly and require only a smallamount of blood for detection of hypoxia-ischemia. Additionally, thereis a need for improvement in determining the oxygen supply status of afetus at partum.

BRIEF SUMMARY OF THE INVENTION

The present invention satisfies at least some of the needs by providinga device and method where LDH, and optionally also AST, ALT, Mg, andlactate, can be analyzed within minutes (or alternatively in seconds) in10 microliters of whole blood. These analyses can be measured togetherwith free haemoglobin to make sure that haemolysis is not present withfalse increased values of LDH as the result.

Embodiments of the present invention include a method of determininghypoxia, especially hypoxia in fetal scalp blood sampled during labor.In such embodiments, the method can comprise the determination of LDH(lactate dehydrogenase) in plasma. It is preferred to determine, inaddition to LDH, one or several markers selected from the groupconsisting of K, Mg, Ca, AST, ALT, and lactate in said fetal scalpblood. Particularly preferred are the combinations of LDH with any of K,Mg, Ca, AST, ALT, lactate. Also preferred is the combination of LDH,lactate, Mg, and AST and/or ALT.

As used throughout, it should be understood that “LDH” and “lactatedehydrogenase” refer to total lactate dehydrogenase, that is, not toisoenzymes thereof.

In the method of the invention scalp blood, in limited amount(preferably about 5-25 μL) is sampled from a fetus through the vaginaduring partum in an environment close to the patient. The sample issubjected to separation of plasma (blood serum) from blood cells, inparticular from erythrocytes, and the plasma is analyzed for LDH and,optionally, for one or more of K, Mg, Ca, AST, ALT, lactate, to presenta result within minutes. As a consequence the medical team may directlyconclude if the child suffers from acute asphyxia, i.e. indicating ifcaesarean section is needed or not. Thanks to the invention a largeamount of the suffering related to hypoxia may be eliminated and also alarge amount of savings may be achieved.

In studies performed by the inventor LDH, AST and ALT in blood samplesfrom children were analysed during the first 24 hours. It was found thatout of 193 tested children. 19 of these children indicated signs ofasphyxia in the form of an Apgar score <7 at the age of five minutes. Bymeans of a ROC curve (see FIG. 1) sensitivity and specificity of 96% forLDH at a cut off value of 1000 U/L could be observed. For HIE thesensitivity is 100% and specificity 96% at the same cut off value. Inother words this means that the main marker according to the method ofthe invention determines 95% (or more) of all children with a truehypoxia correctly at the same time determines 95% of all healthychildren as healthy. AST and ALT presented a sensitivity of 86% and aspecificity of 90% at a cut off value of 55 U/L and 18 U/L respectively.Despite the fact that this study is preliminary it indicates asurprising improvement compared to existing methods of today.

From a cost perspective the results are astonishing. When using methodsof today it is known that it takes about 10 unnecessary caesareansections to find one child with HIE, i.e. in order to find one HIE-childa total of 11 caesarean sections are done and 10 of these beingunnecessary. The sensitivity and specificity of the invention indicatesthat the outcome would be the opposite, i.e. merely one out of tencaesarean sections would be unnecessary. Accordingly society will savemillions and millions and not at least it would result in more childrenbeing able to take advantage of a natural birth. Presented below is atable showing the drastic improvements by the method according to theinvention compared to known methods.

Method LDH AST/ALT CTG pH lactate Sens. HIE 100%  90% 46% 72% Spec. HIE96% 91% 67% 71% Sens. Apgar 91% 86% 90% 57% 66% Spec. Apgar 92% 89% 30%71% 69%

In healthy persons, erythrocytes are known to contain about 150 timesmore LDH than serum. Thus it is important to carry out sampling andseparation in a manner that does not damage erythrocytes to preventtheir LDH content from passing into the plasma. It is also important totake into account the small volumes of scalp blood available foranalysis. In the method of the invention a sample of scalp blood isseparated into plasma and blood cells by a making the plasma passthrough a porous matrix, in this application termed “membrane” on asolid support capable of retaining the blood cells. The plasma isanalysed for LDH in a conventional manner. K, Mg, Ca, AST, ALT, and/orlactate can be analyzed in plasma or in blood; if analyzed in blood, thescalp blood sample has to be divided into a portion from which plasma isobtained for the measurement of LDH, and one or more portions for theanalysis of the other hypoxia marker(s). It is within the ambit of theinvention to use other methods known in the art, such asmicro-centrifugation, microfluidic compact disc technology andmagnetophoresis, for separating plasma from blood cells in the scalpblood sample of the invention.

Methods for analysis of LDH in fluids such as blood plasma are known inthe art and can be adapted to measure LDH in serum obtained from fetalblood; see, for instance, Pinto P V C et al., Clin Chem 15:339-349,1969. Particular useful is the method disclosed in U.S. Pat. No.4,803,159, which is herewith incorporated in this application. Acommercial apparatus for use in the last mentioned method is on themarket (Vitros® DT60 II Chemistry System; Ortho-Clinical Diagnostics,Inc., U.S.A.). Methods for analysis of K, Mg, Ca, ALT, AST, and lactatein blood are routinely used in clinical chemistry and thus within theeasy reach of a person skilled in the art.

According to the invention levels of LDH, K, Mg, Ca, ALT, AST, andlactate are elevated in scalp blood sampled from a fetus through thevagina during partum if the fetus is in a hypoxic state, the standard(baseline) being corresponding levels in a fetus during partum and/orimmediately after partum in the context of an unproblematic birth. Inparticular, levels of LDH, ALT and AST are increased by a factor of two,in particular three or more, in a fetus during partum suffering fromsevere asphyxia, that is, asphyxia which puts the fetus at a higher riskof acquiring HIE.

According to a preferred aspect of the present invention is disclosed amethod of determining hypoxia in a sample of scalp blood during labor,comprising providing a point of care device, using at least one,preferably numerous, markers to within minutes determine if acutehypoxia is at hand.

According to a further preferred variation, the device is additionallyin communication with analyzing means for one or several of K, Mg, Ca,AST, ALT, lactate.

According to a further preferred aspect is disclosed a plasma separationdevice for simple and direct use in a device/method of the invention.

In other aspects, the present invention includes a method of assessinghypoxia at a tissue site of a mammal. In such embodiments, the methodcan comprise the collecting a blood sample from the tissue site, whereinthe blood sample comprises plasma and blood cells followed by separatingthe plasma from the blood cells. The amount of LDH in the plasma isdetermined and the presence of hypoxia at the tissue site is assessedfrom the amount of LDH in the plasma. Methods of assessing hypoxia at atissue site can include analysis of a blood sample from a mammal'sgastrointestinal tract (e.g., a mammal's bowel), analysis of a samplefrom a specific organ (e.g., a mammal's aorta) or cerebrospinal fluidcollected for example by microcatheter, analysis of urine orintraperitoneal fluid, and analysis of a sample from an organ to betransplanted into a mammal in need thereof. Methods of assessing hypoxiaaccording to embodiments of the present invention can allow one topredict the likelihood of brain injury after prenatal asphyxia and theassessment of blood circulation to a mammal's limbs before, during, andafter a medical or surgical procedure.

The present invention will now be explained in more detail by thedescription of preferred but not limiting embodiments thereofillustrated in more or less schematic drawings.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIG. 1 illustrates a ROC curve for sensitivity and specificity;

FIG. 2 depicts a block diagram schematically presenting an embodiment ofthe method of the invention;

FIGS. 3-5 depict sectional views of a first embodiment of a separationdevice in consecutive stages of use in the method of the invention;

FIG. 6 presents an embodiment of capillary device according to theinvention, intended to be used for collecting and testing scalp blood;

FIG. 7 presents a quick test analyzing system according to anotherembodiment of the invention;

FIG. 8 presents an embodiment of disposable card device for a quick testaccording to the invention; and

FIG. 9 presents a diagram of actions taken normally in conjunction withthe invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. As used in the specification, and in the appended claims,the singular forms “a”, “an”, “the”, include plural referents unless thecontext clearly dictates otherwise.

The present invention provides a device and method where LDH, andoptionally also AST, ALT, Mg, and lactate, can be analyzed withinminutes (or alternatively in seconds) in 10 microliters of whole blood.These analyses can be measured together with free haemoglobin to makesure that haemolysis is not present with false increased values of LDHas the result.

In one aspect, the invention comprises a method of determining hypoxia.According to certain embodiments, the method can comprise collecting ablood sample; and determining the total amount of LDH in the blood. Theblood sample can be collected from any mammal or alternatively from anyorgan to be transplanted into a mammal in need thereof.

In another embodiment, the method of determining hypoxia comprisescollecting a blood sample, wherein the blood sample includes plasma andblood cells. Preferably, the plasma is separated from the blood cellssuch that the plasma can be analyzed without the presence of bloodcells. The isolated plasma can be analyzed for the total amount of LDHin the plasma. As such, a determination of LDH can be achieved. Based onthe LDH quantity, or alternatively in conjunction with other prognosticmarkers, a determination of hypoxia can be readily realized.

In one preferred embodiment, the determination of hypoxia includesanalyzing a blood sample to determine the quantity or amount of multipleprognostic markers. In one such embodiment, the method comprisesdetermining the amount of LDH in a blood sample, or preferably theplasma thereof, and determining the amount of at least one additionalprognostic marker selected from the group consisting essentially of K,Mg, Ca, AST, ALT and lactate.

Preferably, the blood sample for analysis is manipulated such that theplasma is separated from the blood cells. In one embodiment, thisseparation can be achieved by use of a semi-permeable membrane orcentrifuge. When utilizing a semi-permeable membrane, the plasmapreferably passes through the membrane and the blood cells are retainedon the membrane. Accordingly, the plasma is for all practical purposesisolated from the bulk of the blood cells. The plasma can then besampled and tested in the absence of blood cells.

According to various embodiments, the sample volume for analysis can begreatly reduced from prior methods. In one embodiment, the volume ofblood for determining hypoxia comprises from 5 μL to 60 μL or from 5 μLto 25 μL, or preferably from about 5 to 15 μL, and in particular 10 μLcertain embodiments, the volume of blood for determining hypoxiacomprises from about 5 μL to 150 μL, or from 10 μL to 120 μL, or from 10μL to 100 μL, or from 10 μL to 80 μL.

Beneficially, embodiments of the present invention allow for thedetermination of hypoxia in a wide variety of circumstances. Forinstance, embodiments of the present invention include, but not limitedto, the determination of hypoxia in blood from a fetal scalp, agastrointestinal tract (e.g., colon anastomosis), specific organs (e.g.,liver and aorta), cerebrospinal fluid from a lumbar drain, and organs tobe transplanted. Additionally, embodiments of the present inventionenable the assessment and/or monitoring of the liver (e.g., in mammal'spotentially suffering from multi-organ dysfunction), peripheral tissue(e.g., related to trauma, sepsis, haemorrhage or extensive surgery),prediction of brain injury after prenatal asphyxia, and monitoring ofperipheral blood circulation of a mammal.

A plasma separation device 1 according to the invention is shown inFIGS. 3-5 in consecutive stages of use in the method. The device 1comprises a generally circular housing in a hydrophobic polymer materialsuch as poly(tetrafluoroethylene). A plasma collection compartment 2 inthe housing has a top opening closed by a plasma separation membrane 11supported by a grid 12 of same or similar material as that of thehousing. The compartment bottom 13 slants towards its center in the formof a wide-angled cone. From a side wall of the compartment 14 extends atubular conduit 15 provided with a valve 16. The conduit 15 extends to asource of negative pressure such as a vacuum pump (not shown). In FIG. 3a 40 μL sample 7 of scalp blood has been deposited on the exterior faceof the membrane 11, the valve 16 being in a closed position. By openingthe valve 16 the compartment 14 is put under negative pressure (FIG. 4);thereby plasma 7′ is sucked into the compartment 14 accumulating on itsslanting bottom 13, the blood cells 7″ being retained by the membrane11. After closing the valve 16 the state shown in FIG. 5 is reached inwhich the pressure in the compartment is equalized with ambient pressureby air sucked through the membrane 11. Alternatively pressureequalization in the compartment 14 can be achieved through conduit 15after stopping the generation of negative pressure, or in any othersuitable way. FIG. 5 illustrates the removal of the plasma sample 7 fromthe compartment 14 by means of an aspirating syringe, the cannula 10 ofwhich has been inserted through the membrane 11. To avoid contaminationof the plasma sample 7 by blood cells on the membrane a separateinsertion port (not shown) can be provided such as, for instance, arubber septum disposed in a separate opening in a top or side wall ofthe device 1. The block diagram of FIG. 2 illustrates the principles ofthe method of the invention.

The compartment may additionally be provided with a second conduitarranged to communicate with a bottom section thereof provided with asecond valve means for controlled emptying of fluid (plasma) accumulatedat the bottom of the compartment. Alternatively, the compartment maycomprise means for determining LDH and other markers in the accumulatedplasma. It is preferred for the membrane to have an area of from 5 mm²to 1000 mm², in particular from 20 mm² to 300 mm².

Scalp blood analysis. The analysis was successfully repeated with scalpblood sampled from a healthy male adult with standard equipment used inclinical routine for pH or lactate measurement. Samples were with andwithout the use of a vaginal tube (to avoid contamination by amnioticfluid in a real life situation). The time from application of the bloodsample to the plasma separation card to the display of the result was 7minutes at average. It is obvious that the time for analysis can beconsiderably shortened, such as to three to four minutes and even less,by using equipment adapted to the method, in particular a separationcard in which the negative pressure applied to vacuum side of themembrane is well controlled and timed, such as by the use of electronicmeans. Thus the use of the method of the invention in the determinationof fetal scalp blood bedside in an emergency situation is perfectlyfeasible.

In the measurements it was found that LDH was substantially higher inscalp blood than in finger capillary blood; LDH: 1044 U/L and 1127 U/Lin scalp blood with and without the use of an amniotic tube,respectively, vs. 400 U/L in finger blood. A divergence between scalpand cord blood similar to that seen for LDH in adult males has beenobserved in respect of glucose and haemoglobin concentration in fetus.It seems that the measurement of LDH in fetal scalp blood is unknown inthe art.

In FIG. 6 there is shown a system according to the invention including adisposable card 2 and an analyzing instrument 3 for performing point ofcare testing, i.e. in an environment nearby the patient, to allow forpresenting a test result within 7 minutes, preferably within 2 minutesand more preferred within seconds. The disposable card 2 is preferablyarranged with a number of different detection cells 20A-20E, as will beexplained more in detail in connection with FIG. 7, but indeed a cardmerely testing LDH (or e.g. two cells for LDH+AST) may in someapplications be sufficient.

In FIG. 6 two consecutive steps according to the method of the inventionare presented. At the upper left-hand side there is shown the first stepwhere new card 2 is provided to be supplied with test blood 7 by meansof a glass capillary device 4, being filled with whole blood amountingto, e.g. about 10 μL. In a consecutive step the glass capillary device 4has been inserted into a compartment 21 of the card 2 to interface theblood sample 7 with the card 2 and the disposable card 2 placed into theinstrument 3, whereby a directly analysis of the blood sample 7 may beperformed as will be explained more in detail in connection with FIG. 7.The card is provided with devices (as known per se, e.g. micro fluidicchannels for distribution of plasma/serum) to allow for plasma 7′ fromthe blood sample 7 to enter into at least one of the detection cell20A-20E, and preferably blood cells in at least one other cell and morepreferred hole blood to at least one other cell. Prior to entering thedetection cells the plasma/serum/whole blood enter into a reactionchamber (26A-26E) where reagents (preferably dried) are deposited.Accordingly reaction will take place prior to the detection. In theinstrument 3 there are positioned optical devices (as known per se, andfor example described in U.S. Pat. No. 4,935,346 (which is herebyincorporated by reference) performing optical measurements. The opticalmeasurement (e.g. spectrophotometry as known per se and described inU.S. Pat. No. 4,803,159 which is hereby incorporated by reference) willdirectly be handled by the processor 31 of the instrument 3 and shown indisplay 32 and/or provided as a data output 33, e.g. on a printed paper.Further the analyzing instrument 3 is preferably provided with bar codereading arrangement 34 enabling reading and processing a bar code 22with a unique code for each disposable card 2. Preferably the analyzinginstrument 3 is enclosed within a housing 35 to make it portable.Connections (not shown) are provided to supply the instrument 3 withnecessary supplies, e.g. power (if not battery operated). In analternative embodiment the instrument may be made smaller by using anexternal processor, e.g. of a laptop making the instrument connectableto laptop, e.g. by means of a USB cable.

In FIG. 7 there is shown an embodiment of an exemplary disposable card 2according to the invention. The card 2 is arranged with five detectioncells 20A-20E, all of them being optical detection cells. The firstdetection cell 20A is for ALT. The second detection cell 20B is for AST.The third detection cell 20C is for LDH total. The fourth and fifthdetection cells 20D, 20E are for lactate and Mg²⁺ respectively. The cardaccording to the shown embodiment has a circular flat shaped body 23,having a diameter which makes it easy to handle, e.g. a diameter withinthe range of 20-120 mm, preferably 40-100 mm. The material of the body23 may be chosen from a wide range, for examplepoly(tetrafluoro)ethylene, polyethylene, polypropylene, polystyrene andsimilar. As schematically presented in FIG. 7 the card 2 is providedwith a chamber 21 adapted to be fitted with a glass capillary device 4supplying a blood sample 7. In connection with the chamber 21, at thebottom thereof there is an interface 23, that in a manner known per sesafeguards further transport of the blood sample, e.g. to the plasmaseparation device 25 (including a membrane and a plasma collectioncompartment, please compare with the membrane 11 shown in FIGS. 3-5). Inbetween the plasma separation membrane 25 and the interface 23 there mayoptionally be provided a sample splitter 24 (as indicated by dottedlines) to provide the possibility to supply whole blood to somedetection cells, e.g. via printed reagents 26 A, B, to detect ALT andAST respectively. A larger amount of the sample of whole blood may thenpossibly be supplied to the plasma separation device 25, e.g. to viaprinted reagents 26 C, D detect LDH total and lactate respectively inthe plasma 7′. Blood cells 7″ obtained after separation may be conductedafter mixing with a printed reagent 26 D to optical cell 20E to detectMg²⁺.

In the following test results will be described wherein differentcombinations of markers are being used in exemplary disposable cardsaccording to the invention.

Lactate dehydrogenase is increasing during hypoxia and is present in allof the body's cells. LDH present in the blood stream indicates hypoxiasevere enough to reduce the blood flow to peripheral organs. This is theonset on leakage of LDH from these cells. By detecting LDH it is notpossible to determine which organ that is suffering from hypoxia. Ifhaemolysis has occurred (rupture of red blood cells will also cause anincrease in LDH, even if it is not by hypoxia). Haemolysis is dividedinto two groups: (1) In vitro, which means that the haemolysis occurwhen taking the sample or at storage in test tubes, and (2) In vivowhich means that the red blood cells within the patient have ruptureddue to illness. An ongoing haemolysis will then give falsely high levelsof LDH, not due to only hypoxia. The half-life (T½) for LDH is dependenton which type of the five isomers of LDH that has leaked out into theblood. LDH1 which is mainly present in the heart, brain and red bloodcells has T½ 120 h while LDH5 mainly appears in the liver and muscleshave T½ at 10 h.

High LDH No Yes Total HIE No 178 6 184 Yes 0 10 10 Total 178 16 194

In our study in newborn infants all the patients with HIE would be foundby using LDH as a marker as well as 178 of 184 healthy patients. Thismeans that no case of hypoxia would be missed and 6 babies would beunnecessarily delivered by caesarean section or by instrumentaldelivery.

High LDH indicates ongoing or a recent episode of hypoxia somewhere inthe body or haemolysis.

AST is an enzyme that is present in many of the organs in the body butis more organ specific than LDH. AST is mainly present in the liver,muscles and red blood cells. AST is, as well as LDH, sensitive towardshaemolysis but not to the same extent.

T½ for AST is 12-15 h for newborns.

High AST No Yes Total HIE No 210 26 236 Yes 0 12 12 Total 210 38 248In our study all the patients with HIE would be found by using AST as amarker as well as 210 of 236 healthy patients. This means that no caseof hypoxia would be missed and 26 babies would be unnecessarilydelivered by caesarean section or instrumental delivery.

High level of AST indicates ongoing or a recent episode of hypoxia inthe liver or muscles or haemolysis.

ALT is a specific enzyme for the liver and is very little affected byhaemolysis.

T½ for ALT is 36 h.

High ALT No Yes Total HIE No 212 28 240 Yes 0 12 12 Total 212 40 252

In our study no baby with brain damage would be missed if ALT is used asa single marker but 28 of 240 healthy patients would be unnecessarilydelivered by caesarean section or instrumental delivery.

An elevated level of ALT indicates an ongoing or an episode of hypoxiain the liver.

The magnesium in the body is to 50% located in the skeleton and 50%inside cells. If an acidosis occurs (lowered pH), for example duringhypoxia, hydrogen ions will move in to the cells. Mg will at the sametime be transported out of the cells into the blood which leads toelevated Mg levels in the blood. For newborns with HIE the Mg levels arelower than for healthy newborns. Mg is more of a marker for acidosisthan cell damage.

An elevated Mg level is an indication of acidosis which is a symptom forhypoxia. A low Mg level is an indication of an episode of hypoxia thatled to brain damage.

At the present stage we have no data on Mg during labor. However; werely on the increase in Mg levels that have been seen in our animalstudies on newborn piglets.

The table below present results based on using LDH+AST as markers.

highAST + LDH No One Both Total HIE No 170 11 3 184 Yes 0 0 10 10 Total170 11 13 194

The findings show that all healthy babies have low levels of AST and/orLDH. Out of the 10 babies with HIE all babies had both high levels ofAST and LDH. Three babies with elevated AST and LDH levels did notsuffer from HIE. AST adds on the effect of using LDH to determine whenthe baby is healthy.

High levels if LDH and AST indicate hypoxia and haemolysis.

The table below presents results based on using LDH+ALT as markers.

highALT + LDH No One Both Total HIE No 163 20 1 184 Yes 0 0 10 10 Total163 20 11 194

The table above shows that if LDH and ALT are analyzed at the same timeall healthy babies have low levels of ALT and/or LDH, Out of the 10babies with HIE all babies had elevated levels of both ALT and LDH. Onebaby with elevated levels of AST and LDH did not suffer from HIE. ALTadds to the effect of using LDH to determine when the baby is healthy.

ALT is also of interest since it theoretically would be able to tell ifthe baby suffered from hypoxia earlier when still in uterus since ALTwill remain in the blood for a long time (T½ is 36 h).

Elevated levels of LDH and ALT indicate hypoxia that has affected theliver (amongst other organs).

The table below presents results based on using LDH+ALT+AST as markers.

highALT + LDH + AST No One Two All Total HIE No 158 20 5 1 184 Yes 0 0 010 10 Total 158 20 5 11 194

If LDH is analyzed together with both AST and ALT and all of the enzymelevels are low no baby with HIE can be found. At an elevation of allthree markers all babies with HIE are found and only one baby isunnecessarily delivered without HIE.

High LDH+ALT+AST is indicating an ongoing hypoxia or hypoxia during theprevious hours. The fact that the half-life of ALT is 24 h longer thanAST and LDH makes this combination a possibility to also give a timeaspect of the hypoxia. For example of LDH+ALT+AST are all high and stillare increasing over the first 24 h after the birth of the baby thatindicates hypoxia appeared in close proximity to the delivery.

In the material used for these calculations actually 23 caesareansections and 22 instrumental deliveries were performed in the group thatshowed no indications on hypoxia (HIE, acidosis or low Apgar score).Here are only the caesarean sections performed due to suspected harm forthe babies are included.

In the example above it is shown that by using LDH in combination withALT only one baby is unnecessarily delivered instead of 45. This gives asaving of 880 000 SEK only in the cost for caesarean sections, the costfor vacuum extraction not included. Vacuum extraction is increasing therisk for injuries for both the mother and the baby which generates costfor the healthcare in combination with increased suffer for the patient.

As seen above AST is not adding much to the method when it comes todetermine weather the baby suffers from hypoxia or not. AST is stilladding information due to the half-time of 12 h. Together with ALT andrepeatedly taking samples after birth these two enzymes are able to giveinformation on when hypoxia occurred. This is of great juridical valuesince obstetricians today have problems to prove if the missed a case ofhypoxia or if the baby already suffered from hypoxia when the womanarrived the hospital for labor.

A conclusion based on information from scientific literature indicatesthat magnesium would add to the knowledge about whether the hypoxia ispresent or was a previous episode.

High levels of LDH+ALT+AST+Mg indicates an ongoing hypoxia severe enoughto affect peripheral organs and give acidosis. High levels ofLDH+ALT+AST but low Mg indicates a recent episode of hypoxia that gaverise to organ damage and also probably affected the brain. Mg is notsensitive towards haemolysis and together with ALT it strengthens thatthe elevated levels of LDH are due to organ damage and not haemolysis.

In the following an example will be described in actual use of themethod and devices respectively according to the invention, assumingthat a patient named Anna arrives to a delivery room to give labor. Thepersonnel starts with controlling the baby's condition by examining theheart frequency with a small piece of equipment on the woman's stomach(fetal heart rate pattern, CTG). At intervals controls with the CTG areperformed to observe possible changes. After eight hours of labor andAnna is opened 8 cm. At a new CTG control it shows that the heart rateof the child is elevated. The midwife calls on the obstetrician on callwho wants to continue to take a sample from the baby's scalp to see ifthere are any other signs on illness.

Anna, who was lying on her side, is asked to move into gynelogicalposition and the obstetrician puts a metallic tube through the vaginaand pushes it to the scalp of the baby. She washes away amniotic fluidand makes a small cut in the baby's scalp (scalp sample). When she seesa drop of blood 7 she takes a capillary tube 4 and takes about 10 μl ofblood. (It is not easy to get blood through the small tube at the sametime as you need it to be clean and work fast. 30-40 μl is quite a largevolume in this setting and that is what is needed for scalp samplesdetecting pH.)

The midwife is next to the obstetrician providing a disposable card 2where the doctor is inserting the capillary tube 4. The card 2 with theblood 7 is then inserted into a detection instrument 3 positioned nearbywhere levels of LDH, AST, ALT and magnesium are analyzed by aspectrophotometer 4. The analyses takes place after the separation ofred blood cells 7″ on the card 2 and the remaining plasma 7′ reacts withreagents 20 A-D on the card. Within minutes (e.g. about two minutes) theresult shows on a display on the instrument. It shows“normal-normal-normal-normal” which tells the obstetrician that none ofthe markers LDH, AST, ALT and magnesium are at elevated levels. Anna iscontinuing the labor by natural means and is later giving birth to ahealthy child.

Later the same night the obstetrician is called into room 3 where Helenais giving birth to her first child. Here as well the CTG is notsatisfactory and a scalp sample is taken. This time the display shows“high-high-high-high” which tells the obstetrician that all of themarkers are at elevated levels. This can only mean that the baby suffersfrom an ongoing hypoxia and a decision to make an emergency caesareansection is made. An alarm to the surgery is made and Helena is rushed tothe room still in her bed. A fast anaesthetic is done while theobstetrician washes her hands and changes to surgical clothes. She isthen doing a cut in the skin and the uterus and a baby is delivered onlyminutes after the alarm.

As described above some of the markers are sensitive towards haemolysis.The influence of heamolysis on the method is not fully investigated atthe present stage, but it seems that it will not drastically change theoutcome in at least some situations. However, if it will show thathaemolysis is appearing frequently during sampling it is foresome tointegrate a marker for haemolysis in the card. The most probablescenario is that we will use free haemoglobin (Hb) as the marker ofchoice. By measuring Hb in plasma information about haemolysis hasoccurred and if it has it will also be possible to see how severe. It isalso possible to take that into account and recalculate the enzymelevels depending on the degree of haemolysis.

In some situations LDH alone or LDH in combination with for examplelactate, ALT, AST, or magnesium, may be enough to judge how the baby isdoing, if so the analysis can be colorimetric (change of colour of thereagents).

In FIG. 8 there is shown an embodiment of alternative testingarrangement, wherein dry chemistry is used to perform the analysis. Anadvantage with testing device 5 as shown in FIG. 8 is that no powersupply at all would be needed. The testing device 5 is arranged withelongated tube formed body/casing 50. Within the elongated body 50 thereis a channel 51 opening up into a front chamber 52 positioned adjacentthe front end of the housing 50. At the other end of the channel 51there is, near the rear end of the housing 50, there is provided apumping device 53. The pumping device 53 is in the form of a resilienthollow body arranged with a check valve 54 at the outlet end thereof.The outlet (via the check valve 54) opens up into a disposal compartment55. At the front end of the housing 50 there is arranged a collar device56 having a centrally positioned hole 57 communicating with the frontchamber 52 via a filter 58, that filters out blood cells. At a distancefrom the front end, forming part of the channel 51, there is a furtherchamber 59 arranged with dry chemistry means 8. Also as a part of thechannel 51 adjacent the pumping device 53 there is arranged a furtherchamber 501 creating a buffer and also providing indication regardingvolume of blood 7 in the channel 51.

The testing device shown in FIG. 8 is intended for use in connectionwith labor. As schematically presented in FIG. 8 scalp of the head 6 ofa child is penetrated to obtain a drop of blood 7 (as in known per se).Thereafter the testing device 5 is introduced via the vagina and putonto the blood sample 7, by positioning the collar 56 around the blooddrop 7. In a next step the pumping mechanism 53 is activated wherein thecheck valve 54 will open up to let air escape from the hollow inner ofthe resilient pumping device 53. When the pumping device 53 is releaseda vacuum will be created (thanks to the resiliency), which iscommunicated to the blood drop 7 via the channel 51, whereby blood willbe sucked into the channel 51 via the filter 58. Accordingly bloodplasma 7′ will enter into the analyzing chamber 59. The pumpingmechanism may be applied a number of times to safeguard sufficient bloodfor the testing, which may be determined once blood is observed in thebuffer chamber 501. Thereafter the testing device 5 is removed and byobserving the colour of the chemical means in the analyzing chamber 59it may be determined if the child is suffering from hypoxia. As is knownper se the dry chemical means 8 may be used to indicate differentmeasures to be taken depending on which colour is presented. Forinstance the following colours could be used to indicate which measureis to be taken. If it shows a green colour no measure should be taken.If a red colour is presented the child should be taken out as quickly aspossible. If yellow colour is presented a new sample should be takenwithin less than 20 minutes.

Clinically the testing device 5 would give the same kind of indicationsas above. It is evident that the use of a capillary tube to collect theblood from the baby's scalp, could also be combined with a slightlymodified testing device 5, but preferably also designed such that whenthe blood is collected a closed system is created within the device 5.This can be achieved in many ways, e.g. by pushing the top with asilicon collar (as shown) against the scalp of the baby or by removingthe device 5 and attach a tight sealed cap on the top (not shown).

We also foresee that the lactate test that is used today can be analyzedin combination with the method of the invention.

In FIG. 9 there is shown a flow chart on when to preferably use themethod and devices respectively according to the invention. As ispresented in the flow chart CTG should preferably be used as a primaryindicator. If the CTG is normal no measure would normally be necessaryto undertake. However if the CTG is abnormal either directly a bloodsample from the child's scalp should be analysed according to theinvention or possibly it may be proceeded by a ST-analysis of the fetalheart.

The invention is not limited by what has been described above but may bevaried within the scope of the appending claims. For instance it isevident for the skilled person that the definition “scalp blood” alsoblood samples collected from other parts of the body may at occasionsfunction to obtain the advantages according to the invention. Samples tobe used with the invention include whole blood, blood plasma and bloodserum.

In another aspect, embodiments of the invention can be beneficially usedin other medical circumstances. In one embodiment, a blood sample iscollected from a location of interest prior to a medical procedure andanalyzed for prognostic markers. Preferably, the plasma and blood cellsare separated from each other prior to determining the respectiveamounts of prognostic markers in the plasma (e.g., total amount of LDHin the plasma). After the medical procedure has been completed, a secondblood sample can be obtained from the point of interest and analyzed inthe same manner as the initial sample. The determination of prognosticmarkers in each sample can determined and compared to assess thepresence of hypoxia.

In various embodiments, multiple prognostic markers are analyzed. Suchembodiments comprise determining total amount of LDH and at least oneadditional prognostic marker in the plasma of both blood samplesselected from the group consisting essentially of K, Mg, Ca, AST, ALTand lactate. Accordingly, the respective amounts of each prognosticmarker in the first and second samples can be compared to identify aproper location for an anastomosis. In one embodiment, the medicalprocedure comprises anastomosis of the gastrointestinal tract.

For example, colorectal cancer is one of the most common tumors in bothgenders. The current treatment involves a surgical procedure where thetumor is removed together with a radical part of the bowel. In themajority of these cases the distal and the proximal ends of the bowelare thereafter put together again. This is referred to as ananastomosis.

As such, certain embodiments comprise a method of determining hypoxiafrom a blood sample collected from a mammal's gastrointestinal tract(e.g. bowel). As such, certain embodiments comprise a point-of-care(POC) method for a quick (e.g. within minutes or seconds as discussedabove) determination of hypoxia-ischemia from a small blood volume.Thus, these embodiments can make it possible to decide if the chosenpart of the bowel is suitable for anastomosis or not.

LDH, AST, magnesium and lactate are markers that increase in blood dueto cellular damage and anaerobic metabolism during hypoxia-ischemia.Before the vessels are extirpated, blood from the bowel can be collectedusing a scalpel (or the like) and a sterile capillary. The capillary canbe inserted into an analysis card described above and analyzed. Theresult can preferably used as a unique patient reference value (e.g., abaseline). After a tumor (and the surrounding bowel) have been removed,a new test can be performed to see if the markers have increased. If so,the area for anastomosis can be relocated closer to the still existingblood supply to minimize the risk for hypoxia-ischemia inducedanastomosis insufficiency and leakage.

In addition to sampling blood from the gastrointestinal tract,embodiments of the present invention can beneficially comprisecollecting blood samples from a specific organ of interest orcerebrospinal fluid of a mammal during a surgical procedure.

For instance, the main reasons for surgery in the thoracoabdominal aortaare aneurysms and dissection. The extensive operation required isassociated with significant morbidity and mortality. Some of the mostcommon complications are neurological injuries. The reason for themajority of these complications is insufficient oxygen and energy supplydue to ischemia. As an example, such operations cause permanentparalysis as a post operative complication in 1-10% of the patientsdepending on the location for surgery on the aorta.

However, embodiments of the present invention comprise a method, whichcan be a POC method, for hypoxia-ischemia detection during surgery inblood from specific organs or cerebrospinal fluid (CSF) through a lumbardrain. As such, a sample from a specific organ of interest is obtainedand analyzed for LDH as described in detail above. In a preferredembodiment, the method for detecting hypoxia includes determining LDHand at least one additional prognostic in the sample selected from thegroup consisting essentially of K, Mg, Ca, AST, ALT and lactate.Preferably, one of the additional prognostic markers determined includeslactate. By way of example, a specific organ of interest can include amammal's aorta.

In another aspect, embodiments of the invention can improve themorbidity and mortality rates in patients after transplantation therapy.One of the key factors impacting morbidity and mortality rates inpatients after transplantation is related to preservation injury ofgrafts, such as the hepatic grafts in a liver transplant. For example,LDH, AST and ALT leakage into the perfusate is an indication of loss ofthe membrane integrity of the liver cells.

In one such embodiment, the method for determining the presence ofhypoxia in an organ to be transplanted into a mammal in need thereof cancomprise collecting a blood sample and analyzing the sample, asdescribed above, for prognostic markers prior to the transplantationsurgery. In one embodiment, the sample is analyzed to determining thetotal amount of LDH and at least one additional prognostic marker in thesample selected from the group consisting essentially of K, Mg, Ca, AST,ALT and lactate. In one preferred embodiment, the organ for transplantcomprises a liver.

Certain embodiments of the present invention can satisfy a need atclinics for identify patients with a risk of developing the criticalcondition multi organ dysfunction. Currently, some countries havedeveloped a team of individuals consisting of physicians and nurses fromthe intensive care unit (ICU) for the purpose of providing a mobile teamthat could come to a medical, not intensive care, ward for assessment ofa patient at risk of multi organ dysfunction. If identified as being atrisk, a patient can be treated and transferred to the ICU. In Australiathe introduction of these mobile teams has reduced the incidence ofcardiac arrest, sudden death due to cardiac arrest, post operativecomplications and days at ICU by as much as 50%.

These mobile teams perform a clinical examination of the lungs, kidneysand circulation to assess the risk of multi organ dysfunction. However,there is no quick reliable method for assessment of the liver ormetabolism. The liver is frequently affected by severe disease and liverenzymes and metabolic acidosis is predictive of mortality.

Accordingly, devices according to embodiments of the present inventionare well suited for use by such teams. Such devices are typically smalland quick enough for a mobile team to bring (e.g in a bag) whenconsulting on a ward. Since LDH, AST and ALT are present in liver cellsand lactate is elevated during metabolic acidosis, these teams can nowadd the assessment of the liver to the other parameters examined byutilizing a device according to the present invention. Thus, devices andmethods according to embodiments of the present invention canpotentiality identify even more patients in risk of severe illness andtherefore save both humanitarian and economical costs.

In another aspect, devices and methods according to certain embodimentsare well suited for use by staff in the intensive care unit (ICU). TheICU is responsible for the most critically ill patients in the healthcare system. Independently, if the patient is taken to the ICU becauseof trauma, cardiac diagnosis, sepsis, haemorrhage or extensive surgerythere is always a substantial risk of hypoxia if the circulation andsaturation are insufficient. Even if the typical parameters arecarefully monitored and abnormalities treated with fluids and drugs,there remains no bedside method capable of providing the ICU staffinformation about the effects of a treatment in the peripheral tissue.

It is known that a substantial increase in serum LDH, AST, ALT andlactate in patient with severe shock is an indicator of a bad outcome(e.g., death) (Hardaway 1981). AST, ALT (and previously also LDH) aremeasured as a clinical routine in many ICUs as a marker of hepaticdamage and lactate is used as a marker of anaerobic metabolism andintestinal ischemia (Juel 2007). However, there remains no device thatcan be operated bedside or provide results in a matter of minutes.

Embodiments of the present invention include a device and a method fordetermining hypoxia bedside, wherein the results are available within amatter of a few minutes at most. Such embodiments include obtaining asample for analysis and determination of LDH. In preferred embodiments,the methods include determining the amount of at least one additionalprognostic marker in the plasma selected from the group consistingessentially of AST, ALT and lactate.

As discussed above, Acute perinatal asphyxia, that is, hypoxia(insufficient oxygen saturation of fetal blood) during or close tobirth, remains an important cause of neurological damage in form ofhypoxic-ischemic encephalopathy (HIE) in newborn infants. Thepathological mechanism of hypoxia-ischemia leading to injuries of theneurons in the brain is biphasic starting with the primary phasedirectly after birth. If the infant is successfully resuscitated thisprimary phase will be followed by a free interval that continues forhours. In two out of seven asphyxiated infants this free interval willbe followed by a secondary energy depletion resulting in delayed celldeath in the child's brain and a clinical picture with seizures (alsoknown as HIE). The free interval offers a possibility to minimize thedelayed cell death by hypothermia treatment (cooling the child's brainto 34.5° C.). However, there is need for a method to predict if a childwill develop HIE and therefore have benefits from hypothermia treatmentand who will not.

Since it is of importance to start the hypothermia treatment as soon aspossible after birth (the neuroprotective effects are best decreased 5.5h after the asphyxia), a quick point of care system for detection ofprognostic markers is of clinical value. Data has been collected fromnewborn infants showing that by determining the amount of LDH togetherwith ALT offers a 100% sensitivity for prediction of HIE with a >96%specificity.

Certain embodiments of the present invention provide a diagnostic toolfor the physician in the clinical workplace and in research aroundhypothermia treatment in neonates subjected to perinatal asphyxia.Further, embodiments of the present invention include a method for thedetermination of hypoxia, in which an individual is capable ofpredicting brain injury after prenatal asphyxia. Such methods includeobtaining a sample for analysis of LDH as discussed in detail above. Inpreferred embodiments, the relative amounts of multiple prognosticmarkers can be determined. Preferably, the blood sample is treated suchthat the plasma is separated from the blood cells and the determinationof the levels of the various prognostic markers is based on an analysisof the plasma alone. In another preferred embodiment, the methodcomprises determining the amount of LDH and at least one additionalprognostic marker in the plasma selected from the group consistingessentially of AST, ALT and lactate. Most preferably, the amount of ALTis one of the prognostic markers determined.

In one method according to embodiments of the present invention, themethod includes providing hypothermia treatment to a mammal in needthereof, wherein the determination of LDH (alone or with otherprognostic markers) in the plasma indicate delayed cell death in amammal's brain.

In yet another aspect, embodiments of the present invention can by usedto assess the status of a mammal's limbs before, during, and aftermedical or surgical treatment. For instance, trauma, fractures andvessel occlusions can affect the circulation to peripheral limbs andmuscles (e.g compartment syndrome). There exists a significantcorrelation between oxygen in ischemic muscle and levels of lactate andLDH is observed (Yamamoto 1988) and lactate is elevated in femoral bloodin patients with peripheral arterial occlusive disease compared tocontrol values (Rexroth 1988). Devices according to embodiments of thepresent invention make it possible to use enzyme and lactate levels todiagnose ischemia of a specific limb and also to assess the effects ofmost treatments.

Additionally, embodiments of the present invention comprise a method fordetermining hypoxia-ischemic by analyzing a sample from a limb ofinterest and determining the total amount of LDH in the plasma.Additional prognostic markers can be quantified at the same time as thedetermination of LDH. This allows an assessment of blood circulation toa mammal's limbs before, during, and after a medical or surgicaltreatment.

The skilled person realizes that a large variety of modifications may beperformed without the use of inventive skill, departing from thedescription above, e.g. the use of glass or some other suitable materialin place of plastic etc.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

1. A method of assessing hypoxia at a tissue site of a mammal, comprising: a. collecting a blood sample from the tissue site, said blood sample comprising plasma and blood cells; b. separating the plasma from the blood cells; c. determining the amount of LDH in the plasma; and d. assessing the presence of hypoxia at the tissue site from the amount of LDH in the plasma.
 2. The method of claim 1, further comprising determining the amount of at least one additional prognostic marker in the plasma selected from the group consisting essentially of K, Mg, Ca, AST, ALT and lactate.
 3. The method of claim 1, wherein the volume of the blood sample comprises from 5 μL to 150 μL.
 4. The method of claim 1, wherein the blood sample is collected prior to a medical procedure and further comprising: a. collecting a second blood sample after a medical procedure, said blood sample comprising plasma and blood cells; b. separating the plasma from the blood cells of the second blood sample; and c. determining the total amount of LDH in the plasma of the second blood sample.
 5. The method of claim 1, wherein the blood sample is collected from one or more of a specific organ, cerebrospinal fluid, urine, or intraperitoneal fluid of a mammal during a surgical procedure.
 6. The method of claim 5, wherein the organ comprises a mammal's aorta.
 7. The method of claim 1, wherein the blood sample is collected from an organ to be transplanted into a mammal of need for said organ.
 8. The method of claim 1, wherein the blood sample is collected prior to transplantation surgery.
 9. The method of claim 8, wherein the organ comprises a liver.
 10. The method of claim 1, wherein the determination of hypoxia allows the prediction of brain injury after prenatal asphyxia.
 11. The method of claim 10, further comprising determining the amount of at least one additional prognostic marker in the plasma selected from the group consisting essentially of AST, ALT and lactate.
 12. The method of claim 10, further comprises providing hypothermia treatment to a mammal in need thereof, wherein the determination of LDH in the plasma indicate delayed cell death in a mammal's brain.
 13. The method of claim 1, wherein determining the total amount of LDH in the plasma allows the assessment of blood circulation to a mammal's limbs before, during, and after a medical or surgical treatment.
 14. A method of determining acute hypoxia in fetal scalp blood, comprising collecting a blood sample during labor and treating the blood sample to look for at least one indication to determine hypoxia characterized in that it comprises the determination of total lactate dehydrogenase (LDH) in plasma obtained from the sample.
 15. The method of claim 14, further comprising the comparison of LDH so determined with LDH determined in plasma obtained from fetal scalp blood sampled during partum from a fetus in a non-hypoxic state.
 16. The method of claim 14, additionally comprising the determination of one or several members of the group consisting of K, Mg, Ca, AST, ALT, and lactate in said scalp blood and/or said plasma.
 17. The method according to claim 14 characterized in that sample is collected on a disposable device including means for separation of plasma and a compartment arranged to perform detection of LDH.
 18. Testing system for performing the method according to claim 14, said testing system comprising a disposable device having a blood sample collecting portion, a plasma separation device and a compartment arranged to indirectly or directly determine the total lactate dehydrogenase (LDH) in the plasma.
 19. Testing system according to claim 18, characterized in that there is a number of detection compartments arranged on the card, wherein preferably each one of said compartments is connected to an upstream compartment with a reagent.
 20. Testing system according to claim 18, characterized in that said body is arranged with a unique code and preferably that said instrument is arranged with a reader.
 21. Testing system according to claim 18, characterized in that said disposable device includes a compartment arranged with dry chemical means for visual detection. 